The present invention relates to nucleic acid molecules which encode a wheat enzyme involved in starch synthesis in plants. This enzyme is a soluble type-1 starch synthase.
The invention furthermore relates to vectors, host cells, plant cells and plants comprising the nucleic acid molecules according to the invention.
Furthermore, there are described methods for the generation of transgenic plants which, owing to the introduction of nucleic acid molecules according to the invention, synthesize starch with altered characteristics.
In view of the increasing importance attributed lately to plant constituents as renewable raw materials, one of the objects of biotechnology research addresses the adaptation of these plant raw materials to the needs of the processing industry. Moreover, to allow renewable raw materials to be used in as many fields as possible, a wide diversity of materials must be generated.
Apart from oils, fats and proteins, polysaccharides constitute the important renewable raw materials from plants. Apart from cellulose, starch—which is one of the most important storage substances in higher plants—takes a central position amongst the polysaccharides. In this context, wheat is one of the most important crop plants since it provides approximately 20% of the total starch production in the European Community.
The polysaccharide starch is a polymer of chemically uniform units, the glucose molecules. However, it is a highly complex mixture of different molecule types which differ with regard to their degree of polymerization, the occurrence of branching of the glucose chains and their chain lengths, which, in addition, may be derivatized, for example phosphorylated. Starch therefore does not constitute a uniform raw material. In particular, amylose starch, an essentially unbranched polymer of -1,4-glycosidically linked glucose molecules, differs from amylopectin starch which, in turn, constitutes a complex mixture of glucose chains with various branchings.
The branchings occur by the occurrence of additional -1,6-glycosidic linkages. In wheat, amylose starch makes up approximately 11 to 37% of the starch synthesized.
To allow suitable starches to be used in the widest possible manner for the widest possible range of industrial needs, it is desirable to provide plants which are capable of synthesizing modified starches which are particularly well suited to various purposes. One possibility of providing such plants is to apply plant breeding measures. However, since wheat is polyploid in character (tetra- and hexaploid), the exertion of influence by plant breeding proves to be very difficult. A “waxy” (amylose-free) wheat was generated only recently by crossing naturally occurring mutants (Nakamura et al., Mol. Gen. Genet. 248 (1995), 253-259).
An alternative to plant breeding methods is the specific modification of starch-producing plants by recombinant methods. However, a prerequisite here is the identification and characterization of the enzymes which are involved in starch synthesis and/or starch modification and of the isolation of the nucleic acid molecules encoding these enzymes.
The biochemical pathways which lead to the synthesis of starch are essentially known. Starch synthesis in plant cells takes place in the plastids. In photosynthetically active tissue, these plastids are the chloroplasts, and in photosynthetically inactive, starch-storing tissue they are amyloplasts.
Important enzymes which are involved in starch synthesis are the starch synthases and the branching enzymes. Amongst the starch synthase, various isoforms have been described, all of which catalyze a polymerization reaction by transferring a glucosyl residue from ADP-glucose to α-1,4-glucans. Branching enzymes catalyze the introduction of α-2,6-branchings into linear α-1,4-glucans.
Starch synthases can be divided into two classes: the starch-granule-bound starch synthases (“granule-bound starch synthases”; GBSS) and the soluble starch synthases (“soluble starch synthases”; SSS). This distinction is not unambiguous in each case since some of the starch synthases exist both in starch-granule-bound form and in solid form (Denyer et al., Plant J. 4 (1993), 191-198; Mu et al., Plant J. 6 (1994), 151-159). Various isoforms have been described, in turn, within these classes for various plant species, and these isoforms differ from each other in terms of their dependency on starter molecules (so-called primer dependent (type II) and primer-independent (type I) starch synthases).
The exact function during starch synthesis has been determined as yet only for the isoform GBSS I, [lacuna] in which this enzyme activity is greatly or fully reduced synthesize an amylose-free (so-called waxy) starch (Shure et al., Cell 35 (1983), 225-233; Visser et al., Mol. Gen-Genet. 225 (1991), 289-296; WO 92/11376), so that a decisive role in the synthesis of amylose starch is assumed to be played by this enzyme. This phenomenon is also observed in the cells of the green alga Chlamydomonas reinhardtii (Delrue et al., J. Bacteriol. 174 (1992), 3612-3620). Moreover, it was possible to demonstrate, in. Chlamydomonas, that GBSS I is not only involved in amylose synthesis, but also plays a role in amylopectin synthesis. Mutants which have no GBSS I activity lack a particular fraction of the normally synthesized amylopectin which contains longer-chain glucans.
The functions of the other isoforms of the starch-granule-bound starch m synthases, in particular of GBSS II, and of the soluble starch synthases are unclear as yet. It is assumed that the soluble starch synthases, together with branching enzymes, participate in amylopectin synthesis (see, for example, Ponstein et al., Plant Physiol. 29 (1990), 234-241) and that they play an important function in regulating the starch synthesis rate.
In wheat, at least two isoforms of the starch-granule-bound starch synthase (60 kDA and 100-105 kDA) and a further isoform which possibly represents a soluble starch synthase (Denyer et al., Planta 196 (1995), 256-265; Rahman et al., Aust. J. Plant Physiol. 22 (1995), 793-803) have been identified at protein level. The presence of several SSS isoforms has already been detected earlier with the aid of chromatographic methods (Rijven, Plant Physiol. 81 (1986), 448-453). A cDNA which encodes wheat GBSS I has already been described (Ainsworth et al., Plant Mol. Biol. 22 (1993), 67 to 82).
Nucleic acid sequences which encode wheat starch synthase isoforms or subsequences of such nucleic acids are known to date from WO 97/45545. cDNA sequences which encode starch synthases other than GBSS I have only been described for peas (Dry et al., Plant J 2 (1992), 193-202), rice (Baba et al., Plant Physiol. 103 (1993), 565 to 573) and potatoes (Edwards et al., Plant J. 8 (1995), 283 to 294) as yet. Soluble starch synthases were identified not only in wheat, but also in a series of other plant species. For example, soluble starch synthases have been isolated to homogeneity from peas (Denyer and Smith, Planta 186 (1992), 609 to 617) and potatoes (Edwards et al., Plant J 8 (1995), 283 to 294).
In these cases, it emerged that the isoform of the soluble starch synthase, which has been identified as SSS III, is identical to the starch-granule-bound starch synthase GBSS II (Denyer et al., Plant J. 4 (1993), 191 to 198; Edwards et al., Plant J. 8 (1995), 283 to 294). The presence of a plurality of SSS isoforms has been described for some other plant species with the aid of chromatographic methods, for example in the case of barley (Tyynelä and Schulman, Physiologica Plantarum 89 (1993) 835-841; Kreis, Planta 148 (1980), 412 to 416). However, DNA sequences which encode these proteins have not been described as yet.
To provide further possibilities of altering any starch-storing plants, preferably cereals, in particular wheat, so that they synthesize a modified starch, it is necessary to identify in each case DNA sequences which encode further isoforms of the starch synthases.